The effects of simvastatin on central dopaminergic systems

The β-actin and D2 receptor mRNA expression of prefrontal cortex in SD rats by RT-PCR after simvastatin treatment at dosage of 1mg, 10mg, 30mg/kg/day.. The β-actin and D1 receptor mRNA e

THE EFFECTS OF SIMVASTATIN ON CENTRAL DOPAMINERGIC SYSTEMS

WANG QING, DENNIS

NATIONAL UNIVERSITY OF SINGAPORE

2005

THE EFFECTS OF SIMVASTATIN ON CENTRAL

August, 2005

ACKNOWLEDGEMENTS

Firstly, I would like to express my deepest appreciation and thanks to my

supervisor Associate Professor Wong Tsun Hon, Peter, Department of Pharmacology,

NUS, for his excellent guidance and immerse support throughout the course of this study

Secondly, I wish to give my great thanks to Prof (Dr.) Tan Benny K.H

Dr Zhu Yi Zhun, and Dr Robert Yang for their proficient guidance and constant

encouragement in many aspects of this project

Thirdly, I am most grateful to my cooperator Dr Wang Ling Zhi, Department

of Pharmacology, for his expert direction and stimulating discussion on the dopamine

transmission measurement My deepest gratitude also goes out to Mrs Ting Wee Lee for

her expert technical assistance and suggestions, with which I have gone smoothly on

many procedures and experiments

I am also greatly thankful to my pal Dr Wang Peng Hua, Department of

Biochemistry, for his encouragement, valuable discussion, and technical support I must

also greatly thank Professor Philip Keith Moore, the Head of Pharmacology Department,

and all of the staffs in the Department of Pharmacology for providing very much

comfortable experimental environment and assistance where necessary

Lastly, I would like to express my most heartfelt gratitude to my parents and

elder brother for their earnest love, constant encouragement and the complete support

throughout my career

This work was supported by a grant (R-184-000-039-112) from the National

University of Singapore

PUBLICATIONS AND MAIN HONORS

1 Wang Q, Wang Ling-Zhi, Boon-Cher Goh, How-Sung Lee, P T.-H Wong Effects

of simvastatin on levels of dopamine and its re-uptake in prefrontal cortex and striatum

among SD rats Submitted

2 Wang Q, Wang P.H, MacLachlan C, and Wong P T.-H (2005) Simvastatin

reverses the downregulation of dopamine D1 and D2 receptor expression in the prefrontal

cortex of 6-hydroxydopamine-induced Parkinsonian rats Brain Research 1045 (1-2),

229-233;

3 Wang Q, Ting W L., Yang H Y and Wong P T.-H (2005) High doses of

simvastatin up-regulate dopamine D1 and D2 receptor expression in the rat prefrontal

cortex: possible involvement of endothelial nitric oxide synthase (British Journal of

Pharmacology 144(7):933-9);

4 Farook JM, Wang Q, Moochhala SM, Zhu ZY, Lee L, Wong PT (2004) Distinct

regions of periaqueductal gray (PAG) are involved in freezing behavior in hooded PVG

rats on the cat-freezing test apparatus Neurosci Lett 354(2):139-42

5 Farook JM, Zhu YZ, Wang Q, Moochhala SM, Lee L, Wong PT (2004) Analysis

of strain difference in behavior to Cholecystokinin (CCK) receptor mediated drugs in

PVG hooded and Sprague-Dawley rats using elevated plus-maze test apparatus

Neurosci Lett 358(3):215-9

Main honors and awards:

1 7th NUS-NUH Annual Scientific Meeting (Oct 2nd -5th, 2003, Republic of Singapore):

The Young Scientist Award (merit)

2 The Third International Congress on Vascular Dementia (October 23rd - 26th, 2003,

Prague, Czech Republic): Oral presentation for 15 minutes

3 The 6th Biennial Meeting of the Asia Pacific Society for Neurochemistry (Feb 4th -7th,

TABLE OF CONTENTS

Acknowledgements i

Publications ii

Table of contents iii

Abbreviations ix

Summary xi

List of tables and figures XiV Chapter 1: Introduction 1

1.1 3-hydroxy-3-methylglutaryl- coenzyme A reductase inhibitors, or statins’ properties and their subtypes

1.1.1 Cholesterol biosynthesis and the inhibition mechanism of statins 2

1.1.2 Several types of statins and their properties 4

1.1.3 Statins and brain diseases 4

1.2 Dopaminergic systems: Dopamine receptors and transmission 10

1.2.1 Dopamine receptors characteristics and their distribution in the brain 10

1.2.2 cAMP the hallmark for the functional measurement of G-protein coupled receptors 17

.1.3 The central dopaminergic systems and neurological disorders 23

1.4 Animal models of Parkinson disease 28

1.5 Nitric oxide synthases and their clinical relevance 30

Chapter 2: Chronic treatment with simvastatin in SD rats 38

2.1 Introduction 38

2.2 Methods 39

2.2.1 Animals and statins pretreatment 39

2.2.2 Serum cholesterol and triglyceride measurement 40

2.2.3.Preparation of RNA from prefrontal cortex and striata of SD rats 40 2.2.4 Reverse transcription-polymerase chain reaction (RT-PCR) 41

2.2.5 Quantification of PCR products 42

2.2.6 Western Blot analysis for D1 and D2 receptors and β-actin 43

2.3 Results 44

2.3.1 Serum cholesterol measurement by kits from ThermoTrace 44

2.3.2 D1 and D2 receptor expression after statin treatment 45

2.4 Discussion 45

2.5 Figures 49

Chapter 3: The effects of Simvastatin on the expression of (e,n,i)NOS, D 1 and D 2

receptors 57

3.1 Introduction: 57

3.2 Methods 59

3.2.1 Animal and statins pretreatment 59

3.2.2 Preparation of RNA from prefrontal cortices and striata of SD rats or mice 59

3.2.3 Reverse transcription-ploymerase chain reaction (RT-PCR) for (e, n, i) NOS (rats), D1 and D2 dopamine receptor (mice) expression 59

3.2.4 Quantification of PCR products 60

3.2.5 Western Blot analysis for eNOS protein 61

3.3 Results 61

3.3.1 NOS mRNA expression measured by RT-PCR in the prefrontal cortex and striatum after simvastatin treatment 61

3.3.2 D1 and D2 dopamine receptor mRNA expression measured by RT-PCR in the prefrontal cortex of eNOS knockout mice 62

3.3.3 eNOS mRNA expression in the prefrontal cortex of SD rats after dopamine receptor D1 and D2 receptors blockade 62

3.4 Discussion 63

3.5 Figures 65

Chapter 4: Functional study of D 1 and D 2 by measuring cAMP levels in the

synaptosomes of the prefrontal cortex in SD rats after the simvastatin treatment 72

4.1 Introduction 72

4.2 Methods 73

4.2.1 Animal and statins pretreatment 73

4.2.2 Different reagents and drugs preparation 74

4.2.3 Preparation of rat prefrontal cortex synaptosomal membranes 74

4.2.4 Synaptosome cAMP measurement after D1 and D2 antagonist or agonist treatment 75

4.3 Results for cAMP measurement 75

4.4 Discussion 76

4.5 Figures 79

Chapter 5: Dopamine content and its re-uptake in the prefrontal cortex and striatum in SD rats after simvastatin treatment 80

5.1 Introduction 80

5.2 Methods 81

5.2.1 Dopamine transmission measurement by HPLC-MS/MS 81

5.2.1.1 Animal and statins pretreatment 81

5.2.1.2 Reagents and standards 82

5.2.1.3 HPLC-MS instrumentation 82

5.2.1.4 Sampling of prefrontal cortex and striatum 82

5.2.1.5 Solid phase extraction 83

5.2.2 Dopamine re-uptake experiment 83

5.2.2.1 Animal and statin pretreatment 83

5.2.2.2 Synaptosome preparation 83

5.2.2.3 Dopamine re-uptake measurement 84

5.3 Results 85

5.4 Discussion 85

5.5 Figures 88

Chapter 6: Effects of simvastatin on dopamine D 1 and D 2 receptor expressions in the prefrontal cortex of 6-hydroxydopamine-induced Parkinsonian rats 93

6.1 Introduction 93

6.2 Methods 95

6.2.1 Parkinson disease animal model—6-OHDA lesioned rats set up 95 6.2.2 Circling behavior and simvastatin pre-treatment 96

6.3 Results 96

6.4 Discussion 97

6.5 Figures 100

Chapter 7: General discussion 105

References

EDTA ethylenediaminetetraacetic acid

GPCR G-protein coupled receptor

HPLC high-performance liquid chromatography

i.p intraperitoneal injection

NaCl sodium chloride

NaOH sodium hydroxide

NO nitric oxide

NOS nitric oxide synthase

eNOS endothelial nitric oxide synthase

iNOS inducible nitric oxide synthase

SD rat Sprague-Dawley rat

SDS-PAGE sodium dedecyl sulphase-polyacryamide gel electrophoresis

SEM standard error of the mean

SKF 82958 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetra-hydro-1H-3-

benzepine hydrobromide Statins HMG-CoA reductase inhibitors or 3-hydroxy-3-methylglutaryl-

coenzyme A reductase inhibitors

SUMMARY

Statins have been widely used clinically to reduce cholesterol level and exert

therapeutic and preventative effects on high risk cardiovascular disorder patients Recent research also showed that statins are beneficial factors not only as anticancer agents but also as preventative agents on some neurological diseases such as Stroke, Alzheimer Disease, and Multiple Sclerosis To date, there is no report about the effects of statins on central dopaminergic systems and their related neurological diseases such as Parkinson’s disease This work is mainly to explore the effects of simvastatin on central dopaminergic systems and its possible mechanism of action

Since statins include two subtypes:lipophilic and hydrophilic First we choose pravastatin and simvastatin (at dosage of 1.0mg/kg/day, 10mg/kg/day and 30mg/kg/day), which represent these two different types respectively, to test if both of them affect the central dopaminergic system to the same extent Our data showed that only simvastatin treatment (10mg/kg/day and 30mg/kg/day) up-regulated D1 and D2 receptors in the prefrontal cortex, but no change in the striatum However, pravastatin treatment did not cause any change in D1 and D2 receptor expression both in the prefrontal cortex and in the striatum Since only the lipophilic simvastatin crosses the brain-blood-barrier (BBB) readily, the results implicated that simvastatin up-regulates dopamine receptors through a central mechanism(s) but not a peripheral one

In contrast, both pravastatin and simvastatin reduced the serum triglyceride but not the cholesterol level in SD rat, indicating that the effect on dopamine receptor expression is probably independent of changes in triglyceride level

In order to verify that dopamine receptor function increased with regulation of receptor expression, the effects of D1 and D2 activation on cAMP levels were studied in cortical synaptosome using immunoassay method D1 receptor activation

up-by chloro-APB (5μM) increased cAMP levels in synaptosomes prepared from the prefrontal cortex of control and simvastatin-treated rats by 88 and 285%, respectively This effect was markedly attenuated by the selective D1 antagonist SCH-23390 (25μM)

D2 receptor activation by quinpirole (5μM) had no effect on the basal cAMP levels in synaptosomes prepared from the prefrontal cortex of control and simvastatin-treated rats, while the same concentration of quinpirole completely abolished the D1 receptor-mediated increase

Concurrent with the up-regulation of dopamine receptor D1 and D2 in the prefrontal cortex, eNOS was found to increase after simvastatin treatment in the prefrontal cortex, but not in the striatum No changes in nNOS and iNOS were observed

in both the prefrontal cortex and striatum However, simvastatin was also shown to regulate dopamine receptor D1 and D2 in the prefrontal cortex of eNOS knockout mice Meanwhile, D1 and D2 receptors antagonists, SCH-23390 hydrochloride and haloperidol have not been found to affect the simvastatin-induced up-regulation of dopamine receptors These results strongly indicated that up-regulation of the dopamine receptors after simvastatin treatment occurs independently of the up-regulation of eNOS

In vitro studies showed that dopamine content decreased in the prefrontal

cortex but increased in the striatum while DA re-uptake in these brain regions remained unchanged

SD rats with unilateral lesion of the medial forebrain bundle by hydroxydopamine showed marked decrease in the expression of dopamine D1 and D2

6-receptors in the prefrontal cortex Simvastatin treatment (10 mg/kg/day for 4 weeks) restored receptor expression to control levels Therefore, these observations suggest that simvastatin may have an effect on cognitive deficits associated with the loss of dopaminergic receptor function in advanced PD

In short, chronic treatment with simvastatin for 4 weeks significantly altered the dopaminergic systems in the prefrontal cortex of SD rats The up-regulation of dopamine receptors expression with concurrent increase in receptor function may have specific implication on cognitive deficits in advanced Parkinson’s disease

LIST OF TABLES/FIGURES

Table 1.1 Pharmacokinetic differences between statins

Table 1.2. Properties of dopamine receptors D1 to D5

Table 1.3 Biochemical properties, regulation, and function of nitric oxide synthases

Figure 1.1 Cholesterol biosynthetic pathway and the inhibition of statins

Figure 1.2 Molecular structures of 3-hydroxy-3-methylglutaryl- coenzyme A

reductase inhibitors

Figure 1.3 D1 receptor structure

Figure 1.4 D2 receptor structure

Figure 1.5 The pathway of GPCR stimulation

Figure 1.6. Dopaminergic pathways in the brain

Figure 2.1. Cholesterol measurement after simvastatin and pravastatin treatment at

Figure 2.4 The β-actin and D2 receptor mRNA expression of prefrontal cortex in SD rats by RT-PCR after simvastatin treatment at dosage of 1mg, 10mg, 30mg/kg/day

Figure 2.5 The β-actin and D1 receptor mRNA expression of prefrontal cortex and striatum in SD rats by RT-PCR after simvastatin and pravastatin treatment at dosage of 30mg/kg/day

Figure 2.6 The β-actin and D2 receptor mRNA expression of prefrontal cortex and striatum in SD rats by RT-PCR after simvastatin and pravastatin treatment at dosage of 30mg/kg/day

Figure 2.7 The β-actin and D1 receptor protein level of prefrontal cortex in SD rats by Western blot after simvastatin treatment at dosage of 30mg/kg/day

Figure 2.8 The β-actin and D2 receptor protein level of prefrontal cortex in SD rats by Western blot after simvastatin treatment at dosage of 30mg/kg/day

Figure 3.1 The β-actin and eNOS mRNA expression of prefrontal cortex and striatum

in SD rats by RT-PCR after simvastatin and pravastatin treatment

Figure 3.2 The β-actin, eNOS, iNOS, and nNOS mRNA expression of prefrontal cortex in SD rats by RT-PCR after simvastatin treatment

Figure 3.3 The β-actin and eNOS protein level of prefrontal cortex in SD rats by Western blot after simvastatin treatment

Figure 3.4 Western blot analysis of eNOS protein in eNOS knockout mice

Figure 3.5 The β-actin and D1 receptor mRNA expression of prefrontal cortex by PCR among control, C57BL mice but treated by simvastatin, and eNOS knockout mice but treated by simvastatin

RT-Figure 3.6 The β-actin and D2 receptor mRNA expression of prefrontal cortex by PCR among control, C57BL mice but treated by simvastatin, and eNOS knockout mice but treated by simvastatin

RT-Figure 3.7 eNOS mRNA expression measured by RT-PCR in the prefrontal cortex of

SD rats after dopamine receptor D1 and D2 were blocked

Figure 4.1 cAMP measurement in the synaptosome of prefrontal cortex after

stimulation with quinpirole, chloro-APB, and SCH-23390 between saline and simvsatatin treated groups

Figure 4.2 cAMP measurement data analysis in the synaptosome of prefrontal cortex

Figure 5.1 Calibration curve for quantification of dopamine in rat brain tissue

Figure 5.2 MRM chromatogram for dopamine (upper) for rat brain and D4-Dopamine (internal standard) (lower)

and1,1,2,2,-Figure 5.3. Dopamine tissue level measurement in the prefrontal cortex and striatum between saline and simvsatatin treated groups

Figure 5.4. Dopamine re-uptake measurement in the prefrontal cortex and striatum between saline and simvsatatin treated groups

Figure 6.1 The β-actin and D1 receptor mRNA expression of prefrontal cortex by PCR among control, 6-OHDA lesioned PD rats, and 6-OHDA lesioned PD rats with simvastatin treatment

RT-Figure 6.2 The β-actin and D2 receptor mRNA expression of prefrontal cortex by PCR among control, 6-OHDA lesioned PD rats, and 6-OHDA lesioned PD rats with simvastatin treatment

RT-Figure 6.3 The β-actin and D1 receptor mRNA expression of striatum by RT-PCR among control, 6-OHDA lesioned PD rats, and 6-OHDA lesioned PD rats with

simvastatin treatment

Figure 6.4 The β-actin and D2 receptor mRNA expression of striatum by RT-PCR among control, 6-OHDA lesioned PD rats, and 6-OHDA lesioned PD rats with

simvastatin treatment

Figure 6.5 The β-actin and D1 receptor protein level expression of prefrontal cortex

by Western blot among control, 6-OHDA lesioned PD rats, and 6-OHDA lesioned PD rats with simvastatin treatment

Figure 6.6 The β-actin and D2 receptor protein level expression of prefrontal cortex

by Western blot among control, 6-OHDA lesioned PD rats, and 6-OHDA lesioned PD rats with simvastatin treatment

CHAPTER 1: INTRODUCTION

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, or statins, have

been widely used in clinical work as one main treatment for most forms of

hypercholesterolemia because of their high efficacy and tolerability than other

lipid-lowering agents In some basic research, it was found that statins reduced the infarct

volume and area of the brain in stroke mice by up-regulating the endothelial nitric oxide

synthase (eNOS) leading to increased blood flow and deceased neurological loss It has

also been showed that nitric oxide (NO) may interact with dopamine receptors by some

unknown mechanisms All of these create one challenging avenue of research for us that

statins are probably intrinsically correlated to dopamine receptors and dopamine content This chapter aims to give an overview of the relationship between simvastatin and central

dopaminergic systems and Parkinson’s disease

1.1 3-Hydroxy-3-methylglutaryl- coenzyme A reductase inhibitors, or statins’

properties and their subtypes

1.1.1 Cholesterol biosynthesis and the inhibition mechanism of statins

The statins are drugs effective in reducing the serum cholesterol levels Hence, medical treatment of hypercholesterolemia has received a major boost from the development of the statins, which inhibit 3-hydroxy-3-methylglutaryl- coenzyme A (HMG-CoA) reductase, a key enzyme in cholesterol biosynthesis The biosynthetic

pathway of cholesterol is detailed in Figure 1.1

As the rate-limiting enzyme, HMG-CoA reductase is the most important and rational target for pharmacological intervention (Goldstein et al 1990; Chorvat et al 1985) Statins act as the competitive inhibitors of HMG-CoA reductase by binding to the active site of this enzyme, thus preventing HMG-CoA reductase from binding with its substrate HMG-CoA (Sit et al 1990)

HMG-CoA reductase is regulated by a receptor system for low density lipoprotein (LDL) in human and other mammals (Brown et al 1986) Its inhibition results in a dramatic reduction of circulating total and LDL cholesterol levels (Hoeg et al.1987) All statins produce compensatory increases in hepatic low-density lipoprotein receptors, resulting in an increased uptake of low-density lipoprotein cholesterol from the blood and the subsequent lowering of circulating cholesterol levels Generally speaking, the clinical effects of statins appear not to differ to a significant extent (Sirtori

et al., 1990), and their physicochemical characteristics may modulate their activities and potential toxicity

Figure 1.1 Cholesterol biosynthetic pathway and the inhibition of statins

statins

1.1.2 Several types of statins and their properties

Examples of statins that are in clinical use include: fluvastatin, pravastatin, atorvastatin, cerivastatin, lovastatin, mevastatin, and simvastatin Their chemical structures are described in Figure 1.2

They can be divided into two groups: hydrophilic and lipophilic The hydrophilic agents include pravastatin and fluvastatin while the lipophilic agents include atorvastatin, cerivastatin, lovastatin, and simvastatin Distribution into the central nervous system (CNS) is dependent on lipophilicity and affinity to p-glycoproteins, which constitute an important efflux mechanism for lipophilic drugs as part of the blood-brain barrier (BBB) For example, by determining the octanol-water partition coefficients (Po/w), Serajuddin et al (1991) showed that lipophilicity increases in the order pravastatin<mevastatin<lovastatin<simvasatain Various pharmacokinetic properties of the drugs are listed in Table 1.1

1.1.3 Statins and brain diseases

In addition to the LDL cholesterol-lowing ability and beneficial effects on cardiovascular diseases, statins have been found to exert beneficial effects on brain diseases such as multiple sclerosis, Alzheimer’s disease, and stroke

Recent epidemiological studies showed that statins might produce a strong reduction in the incidence of Alzheimer’s disease (AD) (Zamrini et al.2004) Meanwhile, increasing interest in cholesterol-lowering drugs has been prompted by recent studies reporting that statins may provide protection against Alzheimer’s disease (Jick et al 2000;

Table 1.1 Pharmacokinetic differences between statins

(Am J Med 2001 Oct 1;111(5):390-400)

Figure 1.2 Molecular structures of 3-hydroxy-3-methylglutaryl- coenzyme A

reductase inhibitors

Wolozin et al, 2000) Statins could substantially reduced the risks of dementia and

Alzheimer’s disease either by delaying its onset, or by opposing specific or general

age-related changes that resulted in cognitive impairment (Jick et al, 2000) Moreover, the

beneficial effects of statins on Alzheimer’s disease were found to be correlated with the

metabolism of amyloid precursor proteins (APPs) and β-amyloid peptides Aβ42 and

Aβ40 (Baskin et al 2003) Fassbender et al (2001) found that simvastatin showed a

strongly reversible reduction of cerebral Aβ42 and Aβ40 levels in vivo in the

cerebrospinal fluid, and in vitro in brain homogenate Simons et al (2001) found that after

treatment with simvastatin in Alzheimer’s patients, these Aβ42 and Aβ40 were reduced

in the CSF In addition, statins also cause changes in tau protein and apolipoprotein E

(ApoE) (Naidu et al 2002; Meske et al 2003)

Statins are also able to reduce the infarct volume in experimental stroke The mechanism of stroke protection appears to be associated with attenuating the

inflammatory cytokine responses that accompany cerebral ischemia, and possessing

antioxidant properties that likely ameliorate ischemic oxidative stress in the brain, and

further improving endothelial function in the absence of significant changes in serum

cholesterol levels (Williams et al 1998; Gerard et al.1997) By administering different

doses of atorvastatin for 2 weeks, Laufs et al (2000) found that atovastatin protected

wild-type mice from cerebral ischemia by up-regulating eNOS mRNA expression in both

aortas and in platelets In his study, atorvastatin was found to reduce the plasma levels of

platelet factor 4 (PF4) and ß-thromboglobulin (ß-TG) in wild type mice but did not alter

plasma levels of PF4 or ß-TG in eNOS knockout mice These findings suggested that

effects of atorvastatin on platelet activity were mediated by eNOS because the plasma

levels of PF4 and ß-TG were not affected by statin treatment in eNOS knockout mice So,

it was concluded that the neuroprotective mechanism of atorvastatin is predominantly mediated by NO-dependent effects of statins on decreased hemostasis of blood and increased blood flow Similar results were obtained by Freedman et al (1997) that NO released from activated platelets markedly inhibited platelet recruitment and thus may limit the progression of intra-arterial thrombosis

NO is an important mediator of vascular homeostasis and blood flow (Palmer

et al., 1987; Radomski et al., 1990; Huang et al., 1995; Furchgott et al., 1980; Ignarro 1990) Huang et al (1996) found that mice that lack the gene for eNOS are relatively hypertensive and exhibit larger cerebral infarctions after middle cerebral artery (MCA) occlusion Administration of NO donors or eNOS substrate increased protection against cerebral ischemia (Dalkara et al., 1994; Zhang et al., 1994; Morikawa et al., 1992) More directly Endres et al (1998) performed similar experiments in eNOS-deficient mice They found that treatment with simvastatin had no effects on cerebral blood flow (CBF), infarct size, or neurological deficits in eNOS-deficient mice Moreover, the percent reduction in regional cerebral blood flow (rCBF) during ischemia was not different between simvastatin and vehicle-injected eNOS deficient mice, and serum cholesterol levels were not significantly affected by treatment with simvastatin The beneficial effects of simvastatin were absent in eNOS-deficient mice indicating that most

if not all beneficial effects are mediated by eNOS Moreover, the levels of nNOS and iNOS mRNA were not significantly different in wild-type and eNOS-deficient mice and were not changed after simvastatin treatment The mechanism of protection may be associated with augmented blood flow owing to up-regulation of eNOS by simvastatin

but may also stem from other known NO-mediated effects such inhibition of platelet aggregation or leukocyte adhesion

1.2 Dopaminergic systems: Dopamine receptors and transmission

1.2.1 Dopamine (DA) receptors characteristics and their distribution in the brain

DA receptors belong to the G-protein-coupled-receptor (GPCR) superfamily, and are divided into two major subtypes: D1 and D2 subfamilies that differ in the coupling to G-proteins, distribution in the central nervous system (CNS), and their pharmacological characteristics With the help of gene cloning procedures, three novel

DA receptor subtypes were characterized over the last ten years: D3, D4, and D5 (Sokoloff

et al., 1990; Van et al., 1991; Sunaharaet al., 1991)

D1 receptor subfamily includes the “classical” D1 receptor (also called the

D1A receptor) and D5 receptor (also called D1B receptor) (Tiberi et al 1991) The difference between them is basically in primary amino acid sequence and anatomical distribution, however; they bind the same receptor-selective agonists and antagonists with similar affinity The D2 receptor subfamily includes the “classical” D2, and also the D3

and D4 receptors These D2-like receptors exhibit a variety of pharmacological, structural and functional similarities (Civelli et al 1993; Gingrich et al 1993; Jackson et al 1994) The structure of the D1 receptor deduced by hydropathic analysis is consistent with that of G-protein coupled receptors (GPCR), with seven hydrophobic domains predicted to traverse the plasma membrane (Sunahara et al., 1991) Among these domains, the amino acids are thought to be in the α-helical configuration Asparagine-

linked glycosylation sites are found on the amino terminal domain and the second extracellular loop (Figure 1.3)

The D1 receptor has a relatively small third cytoplasmic loop, similar to other biogenic amine receptors which are coupled to the stimulatory guanyl nucleotide binding protein Gs (Dohlman et al., 1991; Strader et al., 1989) Activation of D1 receptor results

in elevation of cAMP levels, leading to stimulation of cAMP-dependent protein kinase, inhibition of voltage-dependent K+ channels with subsequent neuronal excitability

D5 receptor is also isolated and cloned from human genomic DNA libraries; its cDNA encodes a 477 amino acid protein that has seven transmembrane domains, a glycosilation site in the N-terminus, a cAMP-dependent phosphorylation site in the third cytoplasmatic loop, and a long residue in the C-terminus, that exhibits high homology to the D1 receptor When D5 receptor is expressed in mammalian cells, it is also functionally coupled to the activation of adenylate cyclase, and GABA receptor-mediated activity through both second messenger cascades as well as through direct receptor-receptor interactions (Yan et al 1997; Liu et al 2000)

Among D1 receptor subfamily, D1 receptor is the most widely distributed central DA receptor In the cerebral cortex, it is largely represented in the prefrontal cortex, anterior cingulated, orbital, insular, piriform, and entorhinal cortex, predominantly V and VI layers In addition to cerebral cortex, D1 receptor is also located

in striatum, accumbens shell, anterior olfactory nuclei, hippocampus, septum, thalamic and hypothalamic nuclei, hindbrain nuclei, and amygdaloid nuclei, etc (Fetsko et al., 2003; Porzio et al., 1999;Blandini et al., 2003) In contrast, the D5 receptor is very restrictedly expressed in the hippocampus, enthorinal and prefrontal cortex, basal ganglia,

and lateral nucleus and hypothalamic nuclei (Ariano et al., 1997; Ciliax et al., 2000; Tiberi et al 1994) Since D1 and D5 receptors are structurally similar to each other, their characteristics are also very similar (Table 1.2)

There are three main types of D2-like receptor: D2, D3, and D4, among which

D2 receptor was the first member in the family to be cloned from rat genomic library Cloned human D2 receptor was found to be 96% identical in amino acid sequence to that

of the rat (Toso et al., 1989; Grandy et al., 1998; Selbie et al., 1989; Stormann at al., 1990) Compared with D1 receptor subfamily, D2 receptor contains a large third cytoplasmic loop a short carboxyl terminal tail and three glycosylation sites in the amino terminal region (see Figure 1.4)

The main structural difference between the D2-like and the D1-like receptor subfamilies is that the C-terminus of the D2-like receptors is rather small and they have the large third cytoplasmic loop between transmembrane regions 5 and 6 and some short carboxyl termini, structural motifs that are characteristic of Gi/o-coupled receptors In contrast to D1 receptor subfamily, activation of Gi/o-coupled receptors lead to inhibition

of adenyl cyclase resulting in a reduction in cAMP level (Senogles 1994; Toso et al., 1989)

The distribution of D2 receptor mRNA has been widely studied Highest levels are observed in neostriatum, especial in larger cells of external globus pallidus, pole and core of nucleus accumbens and olfactory tubercle, as well as in dopaminergic neurons in the substantia nigra pars compacta, ventral tegmental area, midbrain and hindbrain

D3 receptor, which was cloned by Sokoloff et al (1990), shares 52% overall homology and 75% transmembrane homology with the D2 receptor Analysis of D3

Figure 1.3 D 1 receptor structure

receptor mRNA distribution in the brain shows that it is much less abundant than D2

receptor It was found to a greater extent in hypothalamic and limbic nuclei such olfactory tubercle, islands of Calleja, hippocampus, nucleus accumbens, and bed nucleus

of the stria terminalis than in the basal ganglia (Bouthenet et al, 1991; Moine et al., 1995) Minimal expression is also observed in the caudate-putamen, hypothalamus, septum, and geniculate bodies D2 receptor and D3 receptor have similar pharmacological profile (Freedman et al., 1993; Mackenzie et al, 1994; Malmberg et al, 1993; Sokoloff et

al, 1990) such as mediation of adenylate cyclase inhibition

Like D3 receptor, D4 receptor appears to be expressed at a lower level than the D2 receptor Cloning and expression of this receptor was accomplished in several stages (Van Tol et al., 1991) The highest D4 receptor mRNA expression is located in the frontal cortex, midbrain, amygdala, medulla, with lower level in the striatum, olfactory tubercle, and hypothalamus (Van Tol et al., 1991) but it was also found in substantia

nigra pars compacta (Missale et al., 1998) More detailed properties are shown in Table

1.2

Figure 1.4 D2 receptor structure

Table 1.2 Properties of dopamine receptors D1 to D5

1.2.2 cAMP the hallmark for the functional measurement of G-protein coupled receptors

G-protein coupled receptors that bind guanine nucleotides GDP and GTP include three subunits: Gα, Gβ, and Gγ Normally in the inactive state, Gα is bound to GDP As one of the members of G-protein coupled receptors, when ligand such as a D1

receptor agonist binds to D1 receptor, an allosteric change takes place in D1 receptor It causes GDP to be phosphorylated to GTP GTP activates Gα causing it to dissociate from Gβ and Gγ which remain linked as a dimer Activated Gα in turn activates adenylyl cyclase an enzyme in the inner face of the plasma membrane which catalyzes the conversion of ATP into the "second messenger" cyclic AMP (cAMP) There are two main types of Gα subunits: Gαs (stimulatory of adenylyl cyclase), Gαi (inhibitory of

adenylyl cyclase) When Gαs is activated, for example D1 receptor is stimulated by its agonist, Gαs stimulates adenylyl cyclase so that more ATP is converted into cAMP (Figure 1.5) In contrast to activation of D1 receptor, when D2 receptor is combined with its agonist, Gαi will be activated so as to inhibit adenylyl cyclase and lower the level of cAMP (Stoof et al 1981; Stoof et al 1982; Huff 1997)

As an intracellular second messenger that is triggered by hormones or other signaling molecules, cAMP exerts hormonal responses such as the mobilization of stored energy, conservation of water by the kidney, Ca2+ homeostasis, increased rate and force

of contraction of the heart, and also regulates many neural processes

Figure 1.5 The pathway of GPCR stimulation

1.2.3 Dopaminergic pathways and their functions

Dopaminergic neurons, which synthesize and excretes dopamine, are located

in the ventral boundary zone between the midbrain and hindbrain, migrate and form nine cell groups (A8-A16) The three major nuclei substantia nigra, ventral tegmental area (VTA) and hypothalamic nuclei project to nearly all the areas of the brain The projection zones of dopaminergic terminals indict the functional role of dopaminergic neurons and reflect their variability Basically, there are in total four dopaminergic pathways in the brain (Figure 1.6): nigrostriatal pathway, mesolimbic pathway, mesocortical pathway, and tuberoinfundibular pathway (Jucaite 2002)

These nigrostriatal pathway that project from the substantia nigra to subcortical structures (striatum)-caudate nucleus and putamen is the most profused among the four dopaminergic patyways, and constitute about 80% of the dopaminergic systems As part of a system called the basal ganglia motor loop and one of the major dopamine pathways in the brain, it is particularly involved in the production of movement Loss of dopamine neurons in the substantia nigra above 75% produces the main pathological features of Parkinson disease (PD) and leads to a marked reduction in dopamine function in this pathway This pathway is also implicated in tardive dyskinesia, one of the side effects of antipsychotic drugs

The second massive projection is from ventral tegmental area (VTA) which forms two pathways: mesocortical pathway and mesolimbic pathway, which are classically correlated to reward and addiction (Salamone et al., 1996; Spanagel et al., 1999) Historically the mesolimbic and the mesocortical dopamine systems have been considered as the regulatory site for mood, emotion, and the control of visceral activity,

and as such, are the natural sites to explore the role of dopamine in reward The mesocortical pathway connects the ventral tegmentum to the cortex, particularly the frontal lobes As an essential part of the normal cognitive function of the dorsolateral prefrontal cortex (part of the frontal lobe), it is also thought to be involved in motivational and emotional responses Mesocortical pathway is also thought to be associated with the negative symptoms of schizophrenia, which include avolition, alogia and flat affect (lack of emotional response) The mesolimbic pathway links the ventral tegmentum area to the nucleus accumbens in the limbic system This neural pathway is recognized to produce pleasurable feelings and is related to the feelings of desire and reward, particularly because of the connection to the nucleus accumbens (Salamone 1991; Le Moal et al 1991) Thus the mesolimb cortical systems are implicated in reward, reinforcement and addiction Administration of psychostimulants and drugs of abuse has been shown to increase dopamine release in the mesolimbic area, whereas withdrawal of these drugs resulted in a reduction of dopamine (Chiara 1995; Moal et al., 1991; Ramsey

et al., 1992; Wise et al., 1994) Both D1 and D2 receptors are involved in reward and reinforcement behavior, with the agonist stimulating and antagonists inhibiting the behavior (Franklin et al., 1983; Kornetsky et al., 1981) Antipsychotic medications, though poorly understood, disrupt dopamine function in this area implying that especially an excess of dopamine is linked to psychosis and the 'positive symptoms' of schizophrenia (Kane et al., 1994; Sigmundson 1994; Seemanet al., 1995; Seeman et al., 1993; Sokolowski et al 1994; Swerdlow et al 1992) Antipsychotic medication is therefore believed to have its effect by blocking dopamine receptors in this pathway It

is also generally accepted that mesolimbocortical dopamine D1 and D2 receptors play a

role in learning and memory (Sawaguchi et al., 1991; Sawaguchi, T et al., 1994; Arnsten et al., 1995; Levin et al., 1995)

The tuberoinfundibular pathway, which is formed by hypothalamic dopaminergic neurons function mainly to suppress prolactin secretion from the pituitary gland If dopamine is blocked in the tuberoinfundibular pathway, increased prolactin release from the pituitary gland leads to hyperprolactinemia This can cause abnormal lactation (even in men), disruptions to the menstrual cycle in women, visual problems, headache and sexual dysfunction